Endothelial response to fluid shear stress has been strongly implicated as a key factor in the focal development of atherosclerotic lesions in human arteries. The hypothesis of this grant is that the glycocalyx plays a pivotal role in the transduction of mechanical force by vascular endothelium. Our preliminary data as well as recent in vivo studies by others have shown significant changes in the response of confluent endothelium to fluid shear stress when the glycocalyx is removed. In our experiments, removing the glycocalyx compromises the cell's response. Reconstitution of the glycocalyx restores the endothelium's ability to transduce fluid shear forces. Our proposed research will investigate the glycocalyx and its role in mechanotransduction from three interrelated points of view. In Aim 1, we will examine a series of deletions and additions to the glycocalyx and measure the response of the endothelium under different fluid shear stress conditions. The objective is to determine what constituents are active in the response mechanisms and how these might affect the athero-protective and athero-prone regions of the vasculature... In Aim 2, we will measure the mechanical deformation of the glycocalyx directly with new high-resolution optical microscopy based on quantum dot technology, and a comprehensive model of the mechanics of the glycocalyx will be developed based on these measurements. Aim 3 will explore one hypothesized molecular mechanism for the initiation of the biochemical response within the cell: deformation of the transmembrane protein syndecan-4 by mechanical forces leading to cytoplasmic production of shear-related molecular changes. The use of siRNA constructs targeting NO production will allow us to identify the specific proteins participating in the biochemical pathways of transduction for each set of fluid shear stress conditions. Our in vitro results will be extended to in vivo measurements in a mouse model. PUBLIC HEALTH RELEVANCE: The connection between injury to the vascular wall and arterial disease has been known for over forty years, but the mechanisms by which mechanical force causes internal biochemical changes in the artery wall is only now beginning to be understood. This grant will examine the glycocalyx layer on the cells lining the arteries and potentially lead to new insight into the causes of arterial disease.